The present invention relates to a stable microdispersed polyanhydroglucuronic acid (PAGA) and salts thereof, especially suitable for medicinal, pharmaceutical and cosmetic products, as well as to a method of preparing the same. The term polyanhydroglucuronic acid and salts thereof as used herein includes copolymers thereof, especially with anhydroglucose.
Besides proteins, polysaccharides represent the most widespread biopolymers found in the biosphere. As an example, up to 1012 metric tonnes per year of cellulose, a 1, 4 xcex2D-glucane, is synthesized in nature. Other xcex1 and xcex2 glucanes bound e.g. by 1,2; 1,3; 1,4 and 1,6; or 1,2 and 1,4 glycosidic bonds in the main chain, mostly of microbial origin, gain increasing importance with ongoing research in the field. It is the presence of glucuronic acid units in the polymeric chain of the oligosaccharides or polysaccharides that, together with their molar mass and type of the principal glycosidic bond, constitutes the basis of their immunostimulative, antitumourous, anticoagulative, or else haemostyptic effects (cf. Burchard. W. Ed., Polysaccharide, Eigenschaften and Nutzung, Springer Verlag, Berlin 1985, p. 144).
Glucuronoglucanes can preferably be prepared by relatively specific selective oxidation of the primary alcoholic group at C6 carbon atom of the glucopyranosic unit of natural polysaccharides by nitrogen oxides, the C1 aldehydic group of the basic unit being protected by the glycosidic bond.
A variety of methods have been disclosed for preparing glucuronoglucanes and glucuronanes from natural glucanes, using the oxidative effects of NOx either in the gaseous form (Kenyon et al., Ind. Eng. Chem., 41, No 1, 2-8 (1949); DE 0941289; DE 0967144), in nonpolar reaction environment of inert liquids such as hydrogenated hydrocarbons (USSR SU 937462; U.S. Pat. No. 4,347,057; EP 0492990), or in polar environment of aqueous solutions of acids such as HNO3, H3PO4 or their mixtures with HSO4, wherein the NOx are mostly generated directly the oxidation liquor via dosed introduction of reducing substances such as, notably, NaNO2 (GB 709684; CS AO 185366; GB 1593513; Painter J. et al., Carbohydrate Research 140, 61 (1985); Alhaique F., Chim. Oggi 11-15, 17 (1986)), or the reaction environment is created by introducing liquid NOx into aqueous HNO3 (U.S. Pat. No. 4,100,341).
A disadvantage of these known processes relates to the fact that their oxidative effects on the glucane molecule are non-uniform and only relatively specific in that besides creation of carboxyl groups of the uronic type of C6 carbon of the glucopyranosic unit, other types of successive reactions (such as formation of ONO2 and NO groups on C6) and secondary reaction (such as formation of COOH and other oxidised groups on end carbons C1 and C4, and notably on C2 and C3 carbons) do occur. In accord with numerous publications (Kaversneva E. P., Doklady AN SSSR (U.S.S.R.) 78 (3), 481 (1951); Nevell T. P., J. Text. Ind. 42, 91 (1951); Sihtola M. et al., J. Polym. Sci, Part C, (2), 289 (1963); Pastxc3xa9ka M., Chemickxc3xa9 Zvesti (Slovakia) (20), 855 (1966)), extensive testing of polyanhydroglucuronic acids prepared by the action of NOx has led us to the conclusion that, besides carboxyl groups on C6 carbon, several other aldehydes, ketones, and their condensation products are formed that have fundamental influence on the stability of the polyanhydoglucuronic acid product.
It is known that the presence of carbonyl groups can be limited by their back reduction to primary alcoholic groups by means of complex hydrides such as NaBH4 (Charkin S. W. and Brown W. G., J. Am. Chem. Soc. 71, 122 (1949); Mead F. S. M., J. Text, Inst. 46, T 400 (1955)), but this process is quite expensive for industral use due to the cost of the hydrides.
The quality of the product also depends on both the input raw material and the technological method used. Natural glucanes occur in the form of fibres, globules or grains with varying degree of orderliness (crystallinity). Their oxidation and partial degradation due to the effect of NOx does not proceed with the same speed in crystalline and amorphous regions, so that the resulting product represents a mixture of macromolecules oxidised and degraded to various extents which may provide products which are physiologically ineffective and/or have negative effects.
It is evident from the above that the preparation of stable PAGA product having required physical and chemical characteristics, destined for pharmaceutical and cosmetic use, is in no way a simple matter.
In health care practice one often encounters cases of capillary bleeding occurring during injuries or related to surgical interventions. The healing of the wounds frequently depends on attaining rapid homeostasis and creation of coagulum, to especially serve as a protection of the wound against infection. Application of D glucurono-1, 4 xcex2D-glucane, the so-called oxidised cellulose, as a non-toxic resorbable local haemostatics to arrest bleeding from surface injuries or parenchymatous organs, osseous bleeding, and in general wherever use of conventional styptic means may be difficult or slow in functioning and less effective, has proved especially effective in similar cases.
Experience has shown that the product should be stored at temperatures not exceeding 25xc2x0 C., preferably below 10xc2x0 C., protected against direct light. When these conditions are not met, the influence of light and/or elevated temperatures during storage may easily provoke degradation changes due to the instability of secondary reactive groups and, on nitrogen-containing sites. This in turn may be manifested by reduced tissue tolerability, and even virtually exclude application of the conventional product in some pharmaceutical or cosmetic preparations.
In summary, methods of preparing PAGA known thus far are based on oxidative action of NOx on suitable types of polysaccharides of cellulosic or microbial origin (such as scleroglucanes), possibly with subsequent reduction of the content of destabilizing groups via reduction by hydrides, the latter process being, however, relatively expensive and jeopardising the product with simultaneous reduction of the carboxyl group content via reduction of their carbonyls. No method has been found up to now for preparing stable polyanhydroglucuronic acid with broader application scope enabling a better control of the final product characteristics.
Among important disadvantages of the known methods quoted above are non-uniform degree of both oxidation and degradation of individual polysaccharide particles or fibres, non-uniform content of bound nitrogen and other destabilizing sites in the macromolecule, as well as broad distribution of their molecular masses, altogether factors which can result in non-uniformity in resorbtion in the organism on applying the product as a haemostatic or in binding other substances or drugs such as anaesthetics, antibiotics or cytostatics.
In the latter case of active substance-PAGA complexes, the presence of destabilizing groups in this otherwise important biologically degradable carrier brings about inherent instability and changes in properties with time. The same applies to formulations for pharmaceutical or cosmetic use, for which our testing has revealed discoloration with temperature and time, viscosity changes, and even phase separation, whenever unstabilized PAGA prepared by known methods was utilised.
A further deficiency of the known methods lies in the fact that PAGA prepared by NOx oxidation displays closed surface and low values of specific surface area (measured m2.gxe2x88x921) for both fibrillar or particulate material. Whenever final product in powder form is required, the isolated bulk product has to be mechanically disintegrated, in a dry or wet process, which brings about potential contamination by impurities such as metals due to abrasion of production equipment and increases further the production costs.
A last but not least disadvantage of the conventionally prepared PAGA products is that, in contrast to e.g. hyaluronic or algic acids, they do not allow conversion to a range of forms for different applications.
Some of the above deficiencies had been addressed by J Briestensky et al. (CS AAO 242920) who disclose an oxidised cellulose-based sorbent consisting of highly porous non-agglomerated particles of PAGA, and a method of manufacturing the same, involving transformation of oxidised cellulose into a colloidally dispersed system with simultaneous partial hydrolysis followed by coagulation and stabilisation.
However, the above issues relating to the inherent structural non-uniformity of the raw oxidised products and its long-term destabilising effects remain to be unsolved.
There is therefore a need for a method of preparing stable microdispersed polyanhydroglucuronic acid and salts thereof so that the product may be used in medicinal, pharmaceutical and cosmetic formulations.
According to the invention there is provided a method for preparing a product comprising polyanhydroglucuronic acid and/or salts thereof wherein a polyanhydroglucuronic acid-containing material obtained by oxidation with nitrogen oxides is subjected to partial or complete hydrolysis and neutralisation in an aqueous solution of inorganic and/or organic salts and/or bases in the presence of suitable oxidising agents, the hydrolysate undergoing fractional coagulation to form a stable microdispersed/microdispersable product. The term polyanhydroglucuronic acid and salts thereof includes copolymers thereof, especially with anhydroglucose.
This method provides stable polyanhydroglucuronic acid and salts thereof in essentially a single process carried out in a single vessel.
Most preferably the inorganic and/or organic salts and/or bases used for hydrolysis are chlorides, sulphates, carbonates, formates, or acetates of alkali and/or alkaline earth metals, hydroxides of alkali and/or alkaline earth metals, alkylamines, or alkanolamines, in concentrations ranging from 1 to 10xe2x88x923 to 5 mol/l.
In an especially preferred embodiment of the invention the oxidative environment during hydrolysis is established by the presence of oxidising agents selected from one or more of hydrogen, sodium or magnesium peroxide, peroxoacids and their salts, hypochlorites and chlorites.
Preferably the hydrolysate is let to undergo fractional coagulation by a suitable water-miscible organic solvent, the coagulated product is washed, or dehydrated, using a suitable water-miscible organic solvent, and/or converted, in an appropriate manner, for intended subsequent use.
Preferably the procedure is carried out at a pH of from 1 to 12, and preferably, at a temperature of from 0 to 100xc2x0 C.
In a preferred embodiment of the invention the polyanhydroglucuronic acid-containing material is obtained by oxidation of a suitable polysaccharide, such as native or regenerated cellulose or starch.
The invention also provides stable microdispersed/microdispersable polyanhydroglucuronic acid and salts thereof wherever made using the method of the invention. In particular, the invention also provides novel stable microdispersed polyanhydroglucuronic acid and salts thereof containing in their polymeric chain from 8 to 30 per cent by weight of carboxyl groups, at least 80 per cent by weight of these groups being of the uronic type, at most 5 per cent by weight of carbonyl groups, and at most 0.5 per cent by weight of bound nitrogen.
Preferably the product contains at most 0.2 per cent by weight of bound nitrogen in the polymeric chain.
In a preferred embodiment of the invention the molecular mass of the polymeric chain is from 1xc3x97103 to 3xc3x97105 Daltons, most preferably from 5xc3x97103 to 1.5xc3x97105 Daltons.
The content of the carboxyl groups is in the range of from 12 to 26 per cent by weight and at least 95 per cent of these groups are of the uronic type.
In a particularly preferred embodiment of the invention the product contains at most 1 per cent by weight of carbonyl groups. Typically the carbonyl groups are intra- and intermolecular 2,6 and 3,6 hemiacetals, 2,4-hemialdals and C2-C3 aldehydes.
The polyanhydroglucuronic acid and salts thereof may be made up of particles sized from 0.1 to 100 xcexcm and/or fibres of from 5 to 30 xcexcm diameter and up to 30 mm length.
Because neutralisation and refining is carried out in a single operation the process is cost effective.
As the product is in a microdispersed form there is enhanced sorption and greater accessibility for blood. Therefore the biological availability is increased and a rapid onset of haemostasis. We have also observed that the product assists wound healing as a large surface area is presented which is quickly penetrated by body fluids and goes into solution in these fluids. We believe that the product then chemically degrades to achieve more rapid absorption and enhancement of the wound healing process.
The overall homogeneity of the distribution of oxidised groups within the product is increased. Thus, the product has improved reactivity and accessibility to reactive sites for the purpose of binding other substances such as pharmacologically active substances to the product. The average degree of polymerisation is decreased, the distribution of the polymerisation is narrowed and the amount of cellulosic fractions are reduced. This also assists in biodegradation.
The products of the invention are notably free or hydrated aldehydic groups on C2 and C3 carbons of the basic unit, their intra- and intermolecular hemiacetals, intramolecular C2, C6 hemiacetals, intermolecular C2 and C3 hemialdals, and monoketonic groups on C2 and C3 carbons. Presence of even small amounts of these groups may destabilize main glycosidic bonds and result in formation of irritating products, especially in applications in aqueous systems.
In a final stage of the degradation process after oxidation and isolation of the product during its storage, macromolecular products may be formed which are physiologically ineffective or even have irritating or other negative effects on the organism. In addition we have found that equally undesirable from both the physiological and stability standpoint is the content of bound nitrogen, albeit in small concentrations, mostly occurring in the form of nitrosoether or nitrite groups. These groups may undergo scission leading to formation of nitrogen containing acids which in turn may provoke destruction of the PAGA product during storage.
The invention also provides a pharmaceutical or cosmetic composition incorporating stable microdispersed polyanhydroglucuronic acid and salts thereof of the invention.
The invention will be more clearly understood from the following description thereof given by way of example only.
It has been our aim to prepare stable polyanhydroglucuronic acid with controlled physicochemical properties adapted to the intended use, thus reducing or fully suppressing deficiencies of conventional products manufactured as well as broadening the potential scope of applications thereof. This aim is achieved by preparing stabilized microdispersed PAGA with reduced degree of crystallinity, its copolymers with anhydroglucose, and salts thereof, with a high degree of purity.
An important feature of the invention resides in that this microdispersed PAGA, its copolymers with anhydroglucose, and salts thereof, prepared according to the invention, comprising a reduced proportion of the crystalline phase, consists of particles of 0.01 to 1000 xcexcm in size or fibres with 5 to 30 xcexcm diameter and up to 30 mm length, with an open surface, containing in their polymeric chain from 8 to 30 per cent by weight of carboxyl groups, at least 80 per cent of these groups being of the uronic type, and a reduced proportion of destabilizing carbonyl groups, in particular aldehydic ones on C2 and C3 carbons of the basic glucopyranosic unit and condensation products thereof, notably intra-and intermolecular 2,6- and 3,6-hemiacetals, 2,3-hemialdals and C2-C3 aldehydes, as well as of bound nitrogen.
Aiming at suppression of the above mentioned deficiencies, especially of low stability, of the PAGA products manufactured thus far, as well as of deficiencies of known methods of preparing the same, is also the method of preparing according to the invention, which yields stable microdispersed PAGA with easily controllable physicochemical characteristics. An important feature of the process consists in that the raw PAGA product obtained by oxidation of a suitable type of natural glucane and cleared, e.g. by washing, of foreign admixtures is transformed, via action of aqueous solutions of salts such as sodium acetate or carbonate or calcium acetate, chloride or carbonate and/or organic or inorganic bases such as alkyl- or alkanolamines or alkali metals or alkaline earth hydroxides, within an oxidative environment constituted by e.g. organic or inorganic peroxides and/or peroxoacids and salts thereof or hypochlorites or chlorites, into an aqueous colloidal dispersion system, simultaneously provoking hydrolysis of original macromolecular chains of PAGA, oxidation of the destabilizing carbonyl groups in the original PAGA to stable carboxyl groups, and hydrolytic removal of bound nitrogen, whereupon the reaction system is coagulated and stabilised by means of a water-miscible coagulating agent, separated microdispersed PAGA or a salt thereof is washed, isolated, and dehydrated using a water-miscible solvent such as C1 to C4 monohydric aliphatic alcohol, or else first modified by some of known physical or chemical methods and then washed, isolated, and dehydrated in much the same way.